1. Field of the Invention
This invention relates to a chemical process and apparatus involving amino acid derivatization with FMOC using a silicone membrane to extract an undesirable hydrolysis product from the amino acid sample.
2. Description of the Prior Art
Derivatization of amino acids with FMOC is a well known process. FMOC is an abbreviation for 9-fluorenylmethyl chloroformate. This derivatization is performed on a sample prior to testing the sample for amino acids, for instance testing by liquid chromatography to determine exactly what amino acids are present in a sample. This derivatization, i.e., mixing an FMOC solution with an amino acid sample, produces a hydrolysis product called FMOC--OH, which interferes with the peaks of interest in the liquid chromatography process. That is, the derivatization process creates a "ghost peak" in the chromatogram because of the undesirable presence of FMOC--OH in the sample.
FIG. 1 shows the position of the ghost peak 1 caused by FMOC--OH in a conventional chromatogram, with the genuine peaks 2A, . . . , 2G caused by amino acids labeled with the names of the various amino acids. Peak 1 is a ghost because it is not an amino acid peak, but is a by-product of the derivatization reaction, and masks or interferes with important data (i.e., other peaks) in the amino acid analysis. The FMOC derivatization is otherwise a useful process because it is a very sensitive, quick reaction and does not require heating.
A method called liquid-liquid extraction to remove the FMOC--OH contaminant from a sample before testing has been developed in the prior art. In the prior art, the FMOC derivatization process thus includes these steps:
1. Place a sample of amino acid and a quantity of FMOC in a vial and mix.
2. Add pentane (a well known liquid hydrocarbon, C.sub.5 H.sub.12).
3. Mix (i.e., shake the vial) and wait for the mixture to separate into two layers.
4. Pour off the pentane (which is the top layer in the vial) from the lower aqueous layer.
5. Repeat steps 2, 3 and 4 several times.
This process removes most of the FMOC--OH, which is extracted by the pentane. This mixing with pentane and layering is called liquid-liquid extraction, and typically removes about 70% to 80% of the FMOC--OH, usually enough to eliminate interferences from the ghost peak when the derivatized amino acid sample is subjected to liquid chromatography.
FIG. 2 shows the result of a prior art manual pentane extraction, showing a chromatogram with an FMOC--OH peak 1 and a valine amino acid peak 2D, where two extractions were performed. The FMOC--OH peak 1 is 6% of the total peak area. This prior art process has the significant disadvantage that it is a manual method, requiring a skillful technician to observe and pour off the pentane layer.
The prior art process thus has the disadvantages that it is a manual method, and cannot be automated due to the need for visual observation and layer separation; and it is very time consuming (and hence impractical) to prform many extractions, and hence a significant proportion of FMOC--OH is not extracted.
Also well known, but not in connection with amino acid derivatization using FMOC, is membrane extraction. Membrane extraction is known for purposes of extracting trace organic compounds from aqueous samples. Typical of the organic compounds extracted are phenols, caffeine, phenylephrine hydrochloride, and various esters.
R. G. Melcher describes a membrane/flow injection system in "Flow-Injection Determination of Membrane-Selected Organic Compounds", Analytica Chemica Acta 214 (1988) pg. 299-313. Melcher discloses the process of membrane extraction with a continuous (nonporous membrane) such as a silicone rubber membrane. First, an aqueous sample is brought into contact with one surface of the membrane and some of the material to be analyzed is absorbed by the membrane. Second, the material absorbed into the membrane diffuses through the membrane to the second surface of the membrane. Third, some of the material is removed from the membrane by an extracting solution (an extractant) on the second surface of the membrane.
In a dynamic system a constant flow of the sample is maintained on one surface of the membrane and a constant flow of the extracting is maintained on the second surface.
FIG. 3 shows a membrane/flow-injection system as disclosed by Melcher for use in extracting organic compounds such as phenols from an aqueous sample. Shown in FIG. 3 are the extractant reservoir 4, extractant pump 5, membrane 6A in cell 6, detector 8, data recorder 10, carrier (i.e., solvent for the sample) reservoir 12, carrier pump 14, sample inlet 16, six port valve 18, and sample loop 18L in valve 18. Detector 8 is a conventional liquid chromatography detector. The membrane 6A used in cell 6 is silicone rubber tubing. Six port valve 18 is a conventional six port rotary injection valve. The system as shown in FIG. 3 is readily automated. It is typically used for extracting trace amounts of compounds from aqueous samples, so that the extracted compounds (not the aqueous sample) can be subject to testing.